It is a common medical diagnostic procedure to test a blood sample in order to determine the health state of the patient. The hematology panel of tests routinely includes the analysis of red blood cells, their associated morphological indices such as mean corpuscular volume and red blood cell distribution width, hemoglobin, platelets, mean platelet volume and white blood cell differentiation. This panel of tests has previously only been performed on large, expensive and complex instrumentation, but there has been a growing need for less expensive instrumentation that delivers precise and accurate data for all hematology measurements.
In addition, further demands have been placed on instrumentation to analyze more than one species of animals. This need stems from numerous tests that are performed in order to prove the clinical utility of pharmaceutical products, routine analysis of livestock, and the desire to take better care of pets. It has been difficult to generate useful clinical data for multiple animal species from one instrument. This has been a complex problem because the quantity, size, and blood cell membrane permeability has a great deal of variability from one species of animal to the next.
In blood analysis, particular clinical utility has been given to the white blood cells. Within the white blood cell population, many subpopulations exist, five of which are considered predominantly in hematological analysis. These white cell subpopulations include polymorphonuclear neutrophils, which constitute from about 45-80% of the mature white blood cells in a population; monomorphonuclear lymphocytes, which constitute about 15-45% of the white blood cells in a normal mature population; monomorphonuclear monocytes (.about.0-5%); polymorphonuclear eosinophils (.about.0-5%); and polymorphonuclear basophils (0-3%). In many diseases, there is a clinically significant alteration in the normal distribution of these mature cell types. In addition, immature or abnormal cell types are often associated with the disease state. Hematological analysis of white blood cells, including white cell enumeration and flagging of abnormalities, in conjunction with traditional diagnostic techniques, is thus an important tool in the diagnosis and treatment of disease.
Prior to the mid-1970's, white blood cell differential analyses were conducted by manual examination, with a technician viewing blood film slides with the aid of a microscope. Since that time, hematological analysis has been automated, making its use both widespread and commonplace.
While the methodologies for automated analysis vary, most often the enumeration and analysis involves subjecting a diluted sample of whole blood to a lysing reagent which stromatolyzes and therefore eliminates the red blood cell population and simultaneously modifies the cell membranes of the more prevalent white cell subpopulations so that the cytoplasm leeches out, causing differential shrinkage of the different cell types and enabling discrimination and sorting thereof. The size and number of white blood cells in the sample are then detected with the aid of a semi-automated or an automated analyzer, which typically includes a detector adapted to detect the volume (size) or opacity of the white blood cells in the sample by electrical or optical differences. The white blood cells are counted for a period of time sufficient to gather data for analysis, data points are stored in a memory device and then analyzed in an algorithmic processor. The data can then be displayed in the form of a two dimensional or three dimensional histogram.
Heretofore, a number of lysing reagents and reagent systems have been developed which have provided the clinician with the ability to isolate the white cell population from the red cell population of whole blood and to further differentiate the white cell population into smaller subpopulations. Patents considered representative of the art in the field include U.S. Pat. Nos. 3,741,875; 4,286,963; 4,346,018; 4,485,175; 4,529,705; 4,745,071; 4,751,179 and WO88/07187.
U.S. Pat. No. 3,741,875 to Ansley et al describes the use of a cytological fixing agent to kill the blood cells in a fluid sample in order to immobilize the catalytic enzymes contained within the cells. The cytological fixing agent is a concentrated monoaldehyde that must be strong enough (up to 40%) to kill the cells without destroying the activity of enzymes. The methodology of Ansley involves the addition of a specific cytochemical substitute, chromogenic precipitating coupling reagent, and pH buffer after the blood cells have been killed by the monoaldehyde, which causes deposition of an insoluble dye in a specific type of cell containing the immobilized enzyme. The solution containing the dyed blood cells is passed through a photometric counter which gives a count of the dyed cells.
U.S. Pat. No. 4,286,963 to Ledis et al describes a lytic diluent and method for achieving rapid lysis of red blood cells in whole blood. This diluent enhances the ability of automated instrumentation to perform differential determinations of lymphoid and myeloid subpopulations of leukocytes and the quantitative determination of hemoglobin. The lytic diluent employed by Ledis et al is composed of a mixture of at least one quaternary ammonium salt and an aryl substituted short chain alkanol in buffered aqueous medium (pH 3.5 to 5.0). The lytic diluent of this Ledis et al patent is limited in its ability to effect differentiation of the leukocyte population into the two principal subpopulations; namely the lymphoid and myeloid fractions.
U.S. Pat. No. 4,346,018 to Carter et al describes an isotonic blood diluent comprising numerous additives that act as a cell stabilizing mixture of organic buffers, anesthetics, and germicides. Of these ingredients, N-(2-acetamido) iminodiacetic acid (ADA) is a necessary ingredient for stabilizing the size distribution and cellular shape of platelets and erythrocytes. Dimethylolurea is required as a neutral pH in order to afford a measure of stability to the leukocyte membranes.
U.S. Pat. No. 4,485,175, also to Ledis et al, describes a reagent system and method for performance of differential determinations of leukocytes into three subpopulations utilizing automated cell counting equipment, in which the reagent system includes a blood diluent and lysing reagent. The lysing reagent comprises an aqueous mixture of aliphatic quaternary ammonium compounds, which when added to a diluted blood sample under mild conditions of concentration and at a relatively slow rate causes volume modifications to the various subpopulations of white blood cells, enabling a three component separation.
U.S. Pat. No. 4,529,705 to Larsen relates to a reagent for combined diluting and lysing of whole blood cells. The reagent comprises an aliphatic quaternary ammonium salt and at least one anion selected from the group consisting of sulfate, carbonate, formate and acetate anions, which are employed for the purpose of preventing aggregation of platelets in the sample and an alkali metal cyanide for conversion of hemoglobin to a chromagen.
U.S. Pat. No. 4,745,071 to Lapicola describes a reagent system for differential blood analysis including a blood diluent and lysing reagent. The lysing reagent of the `071 patent comprises an aqueous solution of a single aliphatic quaternary ammonium salt and may also contain potassium cyanide as a chromagen forming agent. The preferred quaternary ammonium salt is the dodecyl (C.sub.12) homolog.
U.S. Pat. No. 4,751,179 to Ledis et al describes a reagent system, using saponin as a lysing reagent followed by treatment with a rapidly active cross linking agent such as glutaraldehyde as a fixing reagent. The dialdehyde, preferably glutaraldehyde, must be present in sufficient concentration (0.5-4.0%) to stop the lyric activity of the saponin, and slow acting monoaldehydes, such as formaldehyde, have been determined to be ineffective for preserving the leukocytes in this reaction.
WO88/07187 describes a reagent system enabling differentiation of white blood cells into at least five distinct subpopulations. The PCT application discloses a reagent system comprising a lyric reagent and a companion reagent for the lyric reagent referred to as a "quench". The primary functions of the quench are to retard the activity of the lytic reagent and to restore the ionic balance of the sample subsequent to its treatment with lyric reagent.
Generally, the white blood cell differentials obtained in accordance with the described techniques fall into one of two categories. The first category is a three component screening differential resulting in the isolation and quantification of three fractions of white blood cells including a lymphocyte component, a midregion or monocyte component and a granulocyte (predominantly neutrophilic) component. The granulocyte population usually includes the neutrophils, as well as eosinophils, and possibly basophils, while the midregion often contains monocytes, other mononuclear cells, basophils and eosinophils. Separation among populations is frequently poor, resulting in low correlation between automated and manual differential counts, which is undesirable. Poor separation of subpopulations is further undesirable as it decreases the ability to accurately flag abnormalities.
The second category of white blood cell analyzers is capable of effecting a four or five component separation. Such systems are substantially more complex than the three component differential analyzers, resulting in higher costs to the operator for materials and labor. An illustration of this type of system is described in WO88/07187, a Patent Cooperation Treaty application to Coulter Electronics.
After extensive investigation, it has been determined that the gentle lytic activity achieved by using a single quaternary ammonium salt consisting of either dodecyltrimethyl ammonium salt or dodecyldimethyl ethylammonium salt will indeed preserve infrequent and rare leukocytes, but the identification of abnormalities is hindered because these detergents are non-specific and non-selective to various leukocyte membranes. This non-specificity causes poor definition of these subpopulations, and results in a high degree of overlap, poor correlation to standard methods, and an inability to consistently flag abnormalities.
It has also been determined that the use of a mixture of quaternary ammonium salts as described in previous patents will result in too harsh of lytic activity to provide proper differentiation of subpopulations for semi-automated instruments.
Accordingly, it is an object of the present invention to provide an improved lytic reagent composition which selectively modifies blood cell volume by reacting with the cell membranes to cause red blood cells to rupture and selectively shrinks the white blood cell population into at least three distinct, highly separated components when used in combination with a suitable blood diluent.
Another object of the present invention is to provide a blood diluent capable of providing a stabilized environment to effectively analyze the size distribution of red blood cells, platelets and white blood cells on semi-automated instruments.
A still further object of the present invention is to provide a flexible reagent system for multispecies blood analysis.
A still further object of the present invention is to provide a blood diluent that does not require pH or osmolality optimization for each species in order to be species specific, or a neutral pH to effect a proper leukocyte differential.
Yet another object of the invention is to provide an improved method for semi-automated white blood cell differential analysis.
Further objects and advantages will be appreciated from the following disclosure.